Characterization of a Chimeric Resilin-Elastin Structural Protein Dedicated to 3D Bioprinting as a Bioink Component.

In this study we propose to use for bioprinting a bioink enriched with a recombinant RE15mR protein with a molecular weight of 26 kDa, containing functional sequences derived from resilin and elastin. The resulting protein also contains RGD sequences in its structure, as well as a metalloproteinase cleavage site, allowing positive interaction with the cells seeded on the construct and remodeling the structure of this protein in situ. The described protein is produced in a prokaryotic expression system using an E. coli bacterial strain and purified by a process using a unique combination of known methods not previously used for recombinant elastin-like proteins. The positive effect of RE15mR on the mechanical, physico-chemical, and biological properties of the print is shown in the attached results. The addition of RE15mR to the bioink resulted in improved mechanical and physicochemical properties and promoted the habitation of the prints by cells of the L-929 line.

Graphene Oxide (GO)-Based Bioink with Enhanced 3D Printability and Mechanical Properties for Tissue Engineering Applications.

Currently, a major challenge in material engineering is to develop a cell-safe biomaterial with significant utility in processing technology such as 3D bioprinting. The main goal of this work was to optimize the composition of a new graphene oxide (GO)-based bioink containing additional extracellular matrix (ECM) with unique properties that may find application in 3D bioprinting of biomimetic scaffolds. The experimental work evaluated functional properties such as viscosity and complex modulus, printability, mechanical strength, elasticity, degradation and absorbability, as well as biological properties such as cytotoxicity and cell response after exposure to a biomaterial. The findings demonstrated that the inclusion of GO had no substantial impact on the rheological properties and printability, but it did enhance the mechanical properties. This enhancement is crucial for the advancement of 3D scaffolds that are resilient to deformation and promote their utilization in tissue engineering investigations. Furthermore, GO-based hydrogels exhibited much greater swelling, absorbability and degradation compared to non-GO-based bioink. Additionally, these biomaterials showed lower cytotoxicity. Due to its properties, it is recommended to use bioink containing GO for bioprinting functional tissue models with the vascular system, e.g., for testing drugs or hard tissue models.

Chitosan-based formulation of hemagglutinin antigens for oculo-nasal booster vaccination of chickens against influenza viruses.

Avian influenza viruses (AIVs) and especially highly pathogenic (HP) AIVs of the H5 and H7 subtypes are of both veterinary and public health concern worldwide. In response to the demand for effective vaccines against H5N1 HPAIVs, we produced recombinant protein based on hemagglutinin (HA), a protective viral antigen. A fragment of the HA ectodomain, with a multibasic cleavage site deletion, was expressed in Escherichia coli, refolded, and chromatographically purified from inclusion bodies. Finally, the protein was formulated in Tris-HCl buffer of pH 8.0 or PBS of pH 7.4 to obtain antigens denoted rH5-1 and rH5-2, respectively. The systemic prime and boost immunizations proved that rH5-1 adsorbed to aluminum hydroxide induces anti-H5 HA neutralizing antibodies and protective immune responses against H5N1 HPAIVs in chickens. The present studies were aimed at stimulating immune responses via the mucosal routes using the systemic prime-mucosal boost strategy. Efficacy trials were performed in commercial layer chickens. For systemic and mucosal immunizations, H5 HA antigens were adjuvanted with aluminum hydroxide and chitosan glutamate, respectively. The first dose of rH5-2 was administered subcutaneously, while its second dose was administered subcutaneously, intraocularly, oculo-nasally, or intranasally. rH5-1 was delivered to the subcutaneously primed chickens by the intranasal route. Post-vaccination sera were analyzed for anti-H5 HA antibodies, using homologous ELISA and heterologous FluAC H5 and hemagglutination inhibition tests. Intraocularly and oculo-nasally delivered rH5-2 mixed with chitosan glutamate was capable of stimulating anti-H5 HA IgY antibody responses in the subcutaneously primed chickens; however, it was ineffective when administered by the intranasal route. Efficient intranasal boosting was achieved using rH5-1. The enhanced production of antigen-specific antibodies was reflected in the development of H5-subtype specific and hemagglutination inhibiting antibodies. Conclusively, the subcutaneous prime and oculo-nasal boost vaccination is proposed as the target strategy for future optimization.

Bacterial expression and characterization of an anti-CD22 single-chain antibody fragment.

Single-chain variable fragment (scFv) antibodies are fusion proteins of the variable regions of the heavy and light chains of immunoglobulins connected with a short linker peptide. They possess unique and superior features compared to whole antibodies for immunotherapy of various carcinomas, including hematologic B-cell malignancies. In the presented study we obtained efficient production of the recombinant anti-CD22 scFv in Escherichia coli expression system. The active recombinant protein was successfully recovered from inclusion bodies. Assays were performed to assess the in vitro targeting properties and specificity of the obtained anti-CD22 scFv antibody in the CD22 positive and negative lymphoma cell lines.

Prime-Boost Vaccination With a Novel Hemagglutinin Protein Produced in Bacteria Induces Neutralizing Antibody Responses Against H5-Subtype Influenza Viruses in Commercial Chickens.

The highly pathogenic (HP) avian influenza virus (AIV), H5N1 and reassortant H5-subtype HPAIVs, H5N2, H5N6, and H5N8, cause high mortality in domestic birds, resulting in economic losses in the poultry industry. H5N1 and H5N6 also pose significant public health risks and H5N1 viruses are a permanent pandemic threat. To control HPAIVs, eukaryotic expression systems have traditionally been exploited to produce vaccines based on hemagglutinin (HA), a protective viral antigen. In contrast, we used a bacterial expression system to produce vaccine targeting the HA protein. A fragment of the HA ectodomain from H5N1, with a multibasic cleavage site deletion, was expressed in Escherichia coli, refolded, and chromatographically purified from inclusion bodies. The resulting antigen, rH5-E. coli, was validated in terms of conformational integrity and oligomerization status. Previously, the protective efficacy of rH5-E. coli adjuvanted with aluminum hydroxide, has been positively verified by challenging the specific pathogen-free layer chickens with homologous and heterologous H5N1 HPAIVs. Protection was provided primarily by the H5 subtype-specific antibodies, as detected in the FluAC H5 test. The present studies were conducted to assess the performance of alum-adjuvanted rH5-E. coli in commercial birds. Broiler chickens were vaccinated twice with 25 µg of rH5-E. coli at 2- and 4-week intervals, while the layer chickens were vaccinated with 5- to 25-µg antigen doses at 4- and 6-week intervals. Post-vaccination sera were analyzed for anti-H5 HA antibodies, using homologous ELISA and heterologous FluAC H5 and hemagglutination inhibition (HI) tests. Prime-boost immunizations with rH5-E. coli elicited H5 HA-specific antibodies in all the chickens tested. Two antigen doses administered at 4- or 6-week intervals were sufficient to induce neutralizing antibodies against H5-subtype HAs; however, they were ineffective when applied with a 2-week delay. In the layers, 80% to 100% of individuals developed antibodies that were active in the FluAC H5 and/or HI tests. A dose-sparing effect was seen when using the longer prime-boost interval. In the broiler chickens, 62.5% positivity was achieved in the FluAC H5 and/or HI tests. The trials confirmed the vaccine potential of rH5-E. coli and provided indications for anti-influenza vaccination with respect to the chicken type and immunization scheme.

Expression and purification of recombinant human insulin from E. coli 20 strain.

The number of people with diabetes is estimated to be over 370 million, in 2030 it will increase to 552 million. In Poland, the number of people with diabetes is estimated to be 3.5 million (9.1%). According to the estimates of the International Diabetes Federation, the percentage of patients in the adult Polish population will increase to around 11% over the next 20 years. Despite the appearance of insulin analogues on the pharmaceutical market, insulin delivery is still the most effective method of pharmacotherapy in cases of extremely high hyperglycemia. A new bacterial host strain (Escherichia coli 20) was obtained at the Institute of Biotechnology and Antibiotics and a new pIBAINS expression vector was constructed that provides greater efficiency in the production of recombinant human insulin. In the IBA Bioengineering Department, successful attempts were made to produce recombinant human insulin on a laboratory and quarter-technical scale, and several batches were performed on a semi-technical scale. The production process has been divided into several stages: 1. biosynthesis of insulin in the fermenter, 2. isolation, purification and dissolution of inclusion bodies, 3. protein renaturation, 4. enzymatic reaction with trypsin, 5. multi-stage purification of insulin using low-pressure and HPLC techniques. At each stage of insulin production, qualitative and quantitative analyses were performed to confirm identity and purity. In particular, the molecular weight of insulin, the amount of insulin and the content of protein impurities were studied. The results of these experiments are presented in this work.

Soluble insulin analogs combining rapid- and long-acting hypoglycemic properties - From an efficient E. coli expression system to a pharmaceutical formulation.

The discovery of insulin led to a revolution in diabetes management. Since then, many improvements have been introduced to insulin preparations. The availability of molecular genetic techniques has enabled the creation of insulin analogs by changing the structure of the native protein in order to improve the therapeutic properties. A new expression vector pIBAINS for production of four recombinant human insulin (INS) analogs (GKR, GEKR, AKR, SR) was constructed and overexpressed in the new E. coli 20 strain as a fusion protein with modified human superoxide dismutase (SOD). The SOD gene was used as a signal peptide to enhance the expression of insulin. SOD::INS was manufactured in the form of insoluble inclusion bodies. After cleavage of the fusion protein with trypsin, the released insulin analogs were refolded and purified by reverse-phase high performance liquid chromatography (RP-HPLC). Elongation of chain A, described here for the first time, considerably improved the stability of the selected analogs. Their identity was confirmed with mass spectrometric techniques. The biological activity of the insulin derivatives was tested on rats with experimental diabetes. The obtained results proved that the new analogs described in this paper have the potential to generate prolonged hypoglycemic activity and may allow for even less frequent subcutaneous administration than once-a-day. When applied, all the analogs demonstrate a rapid onset of action. Such a combination renders the proposed biosynthetic insulin unique among already known related formulations.